Molecular and pathogenic characterization of Salmonella enterica serovar Bovismorbificans strains of animal, environmental, food, and human origin in Hungary.

In this study, we characterized 110 strains of Salmonella enterica serovar Bovismorbificans contaminating environment, animals, food of animal origin, and human, to assess their significance along the food chain in Hungary. Additionally, five strains from Germany were tested for comparative purposes. Characterization involved antibiotic susceptibility testing, class 1 integron detection by polymerase chain reaction, plasmid profiling, virulotyping (using virulence gene-specific polymerase chain reactions), and pulsed-field gel electrophoresis. Pathogenic potential of selected strains was tested in orally infected 1-day-old specific pathogen-free chicks. Eighty-two percent of the strains were susceptible to the 16 antibiotics tested, and none of them had class 1 integron. A multidrug-resistant human isolate harbored a bla(SHV5)-type extended-spectrum beta-lactamase gene, first reported in this serotype. All the strains possessed avrA, ssaQ, mgtC, spi4, and sopB genes indicating the presence of Salmonella pathogenicity islands 1-5, respectively, missed the phage-related genes sopE and gipA, but retained the phage-related gene sodC1. An approximately 90 kb large plasmid was characteristic to 80% of the strains, all of which carried the spvC gene. In vivo colonization testing of four selected strains in 1-day-old chicks resulted in significantly reduced liver and spleen colonization ability as compared with the Salmonella Enteritidis control strain, whereas their caecal colonization ability differed less from that of Salmonella Enteritidis. Pulsed-field gel electrophoresis data revealed the dominance of two pulsotypes (C2 and C5) without any specific temporal, geographical, and/or source-related linkages. The results show that Salmonella Bovismorbificans studied here are less invasive than Salmonella Enteritidis, but they may colonize and persist in several animal species and successfully contaminate meat products of different animal origin in Hungary.

Antibiotic resistance and class 1 integron patterns of non-typhoidal human Salmonella serotypes isolated in Hungary in 2002 and 2003.

The antibiotic resistance profiles of 5178 Salmonella strains representing 19 non-typhoidal serotypes isolated from human salmonellosis cases in Hungary in 2002 and 2003 were analysed for resistance to 10 antibiotic agents. The most frequent resistances were to nalidixic acid (Nx), streptomycin (S), tetracycline (Tc), ampicillin (Amp) and chloramphenicol (Cm) (ranging from 27% to 13%). Forty-five percent of the Salmonella Typhimurium strains were multiple resistant and belonged mainly to the definitive phage types 104 and U302. A prevalence of 83-94% of strains of serotypes S. Infantis, S. Hadar and S. Virchow was observed with the NxSTc resistance pattern, sometimes complemented with other resistances. Multiple resistance was uncommon in S. Enteritidis; nevertheless, 20% of the strains, most of which belonged to phage type 4, were nalidixic acid resistant. One strain of S. Typhimurium was found to be resistant to ciprofloxacin. Four S. Typhimurium strains were resistant to cefotaxime and produced extended-spectrum beta-lactamase. Selected isolates were screened for the presence of class 1 integrons by polymerase chain reaction (PCR). Nucleotide sequencing of the PCR products revealed nine different variable regions. One resistance gene was identified in five variable regions (aadA1, aadA2, aadA23, dfrV and pse-1), and four variable regions carried two resistance gene cassettes (aadB-catB3, dhfrI-aadA, dfrA17-aadA5 and oxa-1-aadA1).

Growth and colonization suppression of Salmonella enterica serovar Hadar in vitro and in vivo.

Growth suppression in Salmonella enterica serovar Hadar (S. Hadar) was investigated, in vitro under strict anaerobiosis and in vivo in the intestine of the day-old chicken. Stationary-phase cultures of 20 S. Hadar field strains were tested against each other for growth suppression activity by their ability to suppress the multiplication of low counts of minority cultures inoculated into them as nalidixic acid-resistant mutants. All strains showed profound growth suppression. Four S. Hadar strains were selected and further tested for their ability to suppress growth of S. Enteritidis, S. Typhimurium, S. Virchow and S. Saintpaul. One of the four strains (S. Hadar 18) was randomly selected for further studies. Precolonization of chicken with S. Hadar 18 prevented superinfection with any of the serovars mentioned above. From more than 1000 TnphoA mutants of S. Hadar 18 screened against the parent strain anaerobically in vitro, four were non-suppressive with TnphoA insertions in dapF, aroD, sgaT or tatA. Only the dapF mutant was also non-suppressive in the chicken intestine.

Genes responsible for anaerobic fumarate and arginine metabolism are involved in growth suppression in Salmonella enterica serovar Typhimurium in vitro, without influencing colonisation inhibition in the chicken in vivo.

From a collection of over 2800 Salmonella enterica subspecies Enterica serotype Typhimurium F98 Tn5-TC1 insertion mutants 14 were identified as expressing growth-non-suppressive phenotype under strict anaerobic conditions. Sequence analysis of regions flanking the Tn insertions revealed that most of the selected mutants were defective in genes contributing to the anaerobic fumarate uptake and generation (insertions in dcuA, dcuB and aspA), or to the anaerobic L-arginine utilisation pathway (insertions in STM4467 encoding a putative arginine deiminase, and in between speF encoding ornithine decarboxylase and kdpE coding a response regulator protein). Mutants defective in flagellum synthesis (flhA) were also identified. In contrast to the in vitro results, all the mutants colonised 1-day-old chicks efficiently and suppressed the super-infection of chicks by the parent strain. This clearly indicates that neither of the metabolic pathways mentioned above nor motility play essential roles in lower intestinal tract colonisation.

Analysis of gene cassettes of streptomycin-spectinomycin resistance of Hungarian Salmonella enterica serotype Typhimurium strains.

By PCR using the ant(3")-Ia primer pair the aadA gene was detected in 34 streptomycin- and spectinomycin-resistant Salmonella enterica serotype Typhimurium strains. Out of them 12 belonged to DT104 and 22 to non-DT104 phage type. Using different primer combinations it was demonstrated that this gene was integron-associated in all cases: in the DT104 strains it was generally contained by a 1 kb integron while in the majority of the non-DT104 strains by a 2.05 kb (less often by a 1.9 or 1 kb) integron. In the case of integrons carrying multiple cassettes the cassette containing the aadA gene was located closer to the 3' end of the integron. The aadA genes of DT104 and non-DT104 strains were different: in the former group the aadA2 gene, while in the latter group (constituted by strains of five different phages types as well as unclassifiable and untypable strains) the aadA1 gene could be identified. The RH50/RH51 primer pair described by Collis and Hall (1992) proved to be suitable for rapid discrimination between the aadA1 and aadA2 genes on the basis that the RH51 primer bound exclusively to the aadA2 gene.

Antimicrobial resistance and genetic characteristics of integron-carrier shigellae isolated in Hungary (1998-2008).

Antimicrobial susceptibility, integron carriage, genetic relationship and presence of some important virulence genes of the integron-carrier strains of Shigella sonnei (n = 230) and Shigella flexneri (n = 22) isolated from stool samples of patients in Hungary between 1998 and 2008 were investigated. Sixty-seven per cent (168/252) of the strains were resistant to sulfamethoxazole/trimethoprim (SxT) followed by streptomycin (S, 47%), ampicillin (A, 32%) and tetracycline (Tc, 28%). Thirty-six per cent (90/252) exhibited multidrug resistance, mostly showing SSxTTc or ASSxTc, ASSxTTc resistance patterns. An S. sonnei strain of imported origin was resistant to cefotaxime and harboured a blaCTX-M-55-type extended-spectrum ß-lactamase gene. Altogether 33% of the S. sonnei (n = 75) and 14% of the S. flexneri (n = 3) strains had either class 1 or class 2 integrons or both. The variable regions encoded aadA1 or dfrA1-aadA1 genes for the class 1 and dfrA1-sat2-aadA1 or dfrA1-sat2 genes for the class 2 integrons. Pulsed-field gel electrophoresis analysis revealed that those strains that have different integron types represented different genetic clusters. The Shiga toxin (stx1) gene was identified in one S. sonnei strain and the cdtB gene was detected in an S. flexneri strain. The results reveal the high incidence of antibiotic resistance among Shigella isolates and the presence of the stx1 gene in S. sonnei and the cdtB gene in S. flexneri. The genetic diversity of Shigella spp. isolated recently in Hungary was also demonstrated.

Emergence of multidrug-resistant clones of Salmonella Infantis in broiler chickens and humans in Hungary.

OBJECTIVES: The characterization of a Salmonella Infantis strain collection that was set up from isolates of animal and human origin obtained in Hungary in recent years. METHODS: All isolates were phage typed. Antimicrobial resistance was tested by the disc diffusion method, while the presence of the antimicrobial resistance genes and class 1 integrons was investigated by PCR. Genetic relatedness of the isolates was tested by PFGE and plasmid profiling. RESULTS: The majority of the isolates representing different parts of Hungary are characterized by phage types 213 and 217 and the nalidixic acid-streptomycin-sulphonamide-tetracycline resistance type. They harbour a class 1 integron with an aadA1 gene in the 855 bp variable region, a tet(A) gene, a >168 kb plasmid and 66% of them represent one genetic clone as determined by XbaI PFGE fingerprinting. CONCLUSIONS: It seems that broiler chickens constitute a reservoir for one large and a few smaller multidrug-resistant Salmonella Infantis clones in Hungary, which might have spread to humans through chicken meat.

Class 1 integrons and their conjugal transfer with and without virulence-associated genes in extra-intestinal and intestinal Escherichia coli of poultry.

Multiple-drug resistance in enteric bacteria is frequently associated with integrons. To determine whether integrons may play a role in avian pathogenic Escherichia coli, isolates of extra-intestinal (n = 27) and intestinal (n = 40) E. coli from dead chicks and turkey poults were analysed for the presence of class 1 integrons and of the virulence-associated genes iss, tsh and colV. Eleven extra-intestinal strains possessed a 1.0 kb class 1 integron with a variable region of aadA1 and were resistant to tetracycline. These traits were indicative of the presence of the Tn21 transposon, which was confirmed by polymerase chain reaction. All extra-intestinal strains had the colV, iss and tsh genes, but none of these genes were cotransferred with the 1.0 kb integron when conjugal transferability was tested. The integron content of the intestinal strains showed considerable variability: one or two of four different class 1 integrons, which varied from 1.0 to 2.4 kb in size, were detected in the 11 strains. The aadA7 gene of the 1.0 kb integron, the dfrA1-aadA1 genes of the 1.6 kb integron and the folA-catB3-aadA5 genes of the 2.4 kb integron were identical to those described by other workers. However, the orfIN682-dhfrV-orfD gene cassette arrangement of the 1.5 kb integron of an intestinal strain of serogroup O5 had no similarity to any previously reported integrons. Conjugal transfer of the 1.6 and 2.4 kb integrons was successful, and in a serogroup O33 intestinal E. coli strain the iss gene was apparently cotransferred with a 1.6 kb integron. The 1.0 and 1.5 kb integrons were not transferable. Our data suggest that intestinal E. coli strains of poultry may be a reservoir for emerging multiresistant strains of avian pathogenic E. coli.